Project Summary a-catenin is the key linker between the cadherin/catenin complex (CCC) and the actin cytoskeleton, and must both withstand and respond to changes in tension at adherens junctions (AJs). Understanding how ?-catenin and its function interactors behave under tension has widespread implications for understanding and treating defects during embryonic development, for modulating cell behavior in tissue engineering applications, and for diagnosing and treating metastatic tumors. This proposal seeks to use an innovative combination of molecular structural analysis, molecular biophysics, and in vivo approaches, using the C. elegans embryo as a model system, to investigate key domains in ?-catenin required for its attachment to junctions under tension, and to investigate novel tension-sensitive functional interactors with ?-catenin, SRGP- 1/srGAP and TES-1/Tes. This project has four major components: (1) Role of specific N-terminal domains in mediating the ?-/?-catenin interaction. Using a new crystal structure, we will investigate how individual helices allow ?- and ?- catenin to bind to one another. We will also test the in vivo importance of two conserved residues in HMP-2/?-catenin for its association with HMP-1/?-catenin. (2) Role of the M domain in regulating F-actin binding. The internals domain(s) that regulate ?-catenin binding to F-actin are unknown. Our novel preliminary data indicate that the affinity of HMP-1/?-catenin for F-actin is negatively regulated by an internal region that includes part of the M domain. We will test the importance of this region using biochemical and in vivo structure-function approaches. We will also test whether M domain stability is functionally important for negative regulation of actin binding, and whether this region regulates a tension-dependent increase in binding affinity of HMP-1 for F-actin using innovative single-molecule biophysical approaches. (3) Role of the M domain in recruitment and activation of the novel binding partner, SRGP-1/srGAP: SRGP-1 is a novel HMP-1/ ?-catenin M domain binding partner that is recruited to junctions under tension in vivo via its C terminus. We will use single- molecule biophysical approaches to test whether the interaction of SRGP-1/srGAP with HMP-1 is tension-dependent. We will also assess how HMP-1-dependent recruitment activates crucial functions of SRGP-1 at junctions and assess the role of these identified functions. (4) Role of TES-1/Tes and ZYX-1/zyxin in tension-dependent strengthening of adherens junctions. Using functional genomics, we identified the single Tes homologue, TES-1, and its binding partner, ZYX-1/zyxin, as component of AJs. ZYX-1 directly binds HMP-1 and coIPs with TES-1, leading to a testable model of ZYX-1 recruitment to mature junctions via HMP-1, which then recruits TES-1 to reinforce attachments to F-actin at mature junctions. We will also extend the ?-catenin interactome through characterizing extragenic suppressors of a weak HMP-1/?-catenin mutant. As a result of these studies, we will gain new insight into how adherens junctions are able to withstand and respond to tension in a living organism, a process crucial for diverse cellular events during human development and disease.